JP2013106924A - Temperature-responsive chondrocyte-containing fluid formulation for cartilaginous tissue regeneration, production method, and utilization method of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cartilaginous tissue regeneration medial formulation capable of increasing phenotypic expressions of chondrocytes thereby suppressing inflammation of a recipient site.SOLUTION: In a temperature-responsive chondrocyte-containing fluid formulation, hyaluronic acid grafted to a polymer whose hydration force changes in the temperature range of 0-80°C is dissolved in saline, and 1×10pieces to 1×10pieces of cells are dispersed per ml of the formulation.

Description

  The present invention relates to a preparation for cartilage tissue regeneration useful in fields such as biology and medicine, and also relates to a production method and a utilization method thereof.

  Japan has an aging society and the average life expectancy is the highest in the world. People's hope has come to focus on quality of life (QOL) to live better than just prolong life. One of the attentions is motor dysfunction. Arthritis that causes motor dysfunction includes various diseases, but in the United States, over 70 million patients in the United States complained of some arthritis or chronic joint symptoms in 2002. This is expected to reach 1 in 3 adults and double by 2020. It is the second most common cause of problems in daily life after heart disease, and costs 86.2 billion dollars a year. Of these, osteoarthritis is one of the main causes, reaching over 20 million people over the age of 45. In Japan, the incidence of osteoarthritis is high, with a prevalence of 30% at the age of 45-65 and 63-85% at the age of 65 and over. The total number of patients in Japan has reached 1 million, 900,000 every year. It is believed that new patients will occur. Although musculoskeletal diseases such as osteoarthritis and the like are unlikely to be directly life threatening unlike organ diseases, they deprive human hands and feet and significantly lower their QOL. These musculoskeletal diseases are expected to increase with the aging of the future, and human and social losses due to such disabilities are extremely large.

  Most of these musculoskeletal diseases are caused by inflammation or damage of cartilage tissue and bone tissue. Currently, in the case of severe diseases, artificial joints made of metal and ultra-high molecular weight polyethylene are used for the treatment. However, it wears over 10 years after implantation and the wear powder causes various undesirable biological reactions. In order to solve these problems, studies have been made to improve the wear resistance, but a limit is predicted in the wear resistance. As a new solution, the treatment of cartilage tissue and bone tissue using tissue regeneration engineering technology has attracted attention. As a method for this treatment, a method of transplanting cartilage cells or bone cells cultured in the affected area, and cartilage tissue and bone tissue produced therefrom is considered.

  In 1994, Brittberg et al. Reported a treatment method in which articular cartilage tissue was collected from the non-overweight part of the joint, and the isolated cartilage tissue cells were cultured and transplanted to the osteochondral full-thickness defect part of the overweight part (Non-patent Document 1). Since then, it has been approved by the FDA in 1997, has been commercialized, and has already counted more than 20,000 cases worldwide. The medium- to long-term results of 219 cases after 2 to 10 years were good, and functional improvement was recognized in 89% (Non-patent Document 2). On the other hand, there were reports of deaths due to bacterial infection after transplantation in 2002, and 41 cases of postoperative infections were found in the CDC survey. I was reminded that there are case examples and there are some issues that should be treated carefully.

  In Japan, cartilage tissue was prepared by tissue engineering using chondrocytes separated from non-overweight articular cartilage and bone marrow-derived mesenchymal stem cells, and clinical application has been started for osteoclastic full-thickness cases Yes. However, these clinical application examples are traumatic osteochondral injury and transected osteochondritis, and the range of cartilage defect was originally limited to only small cases (Patent Documents 1 to 3). At present, since the treatment results of artificial joint replacement are stable, it can be said that the treatment of osteoarthritis with extensive cartilage and bone tissue degeneration and partial defects has not been completed.

  Cell culture is usually performed on the surface of a glass or a synthetic polymer subjected to various treatments. For this purpose, for example, various containers subjected to surface treatment using polystyrene as a material, for example, γ-irradiation, silicone coating, and the like are widely used as cell culture containers. Cells cultured and proliferated using such a cell culture container are peeled and collected from the container surface by treatment with a protease such as trypsin or a chemical. However, when recovering cells grown by chemical treatment as described above, the treatment process becomes complicated, the possibility of contamination is increased, and the grown cells are altered or damaged by chemical treatment. Disadvantages have been pointed out that there are cases where the original function of cells is impaired.

  In order to overcome such drawbacks, several techniques have been proposed so far. Among them, in Patent Document 4, in particular, an anterior ocular segment-related cell is cultured on a cell culture substrate in which a temperature-responsive polymer having an upper or lower critical dissolution temperature in water of 0 to 80 ° C. is coated on the substrate surface. However, if necessary, it is possible to produce a cell sheet with sufficient strength by layering the cultured cell layer by a conventional method and peeling the cultured cell sheet simply by changing the temperature of the culture substrate. It was. In addition, this cell sheet also retains a basement membrane-like protein, and the engraftment to the tissue is clearly improved as compared with the above-mentioned dispase-treated one. In Patent Document 5, cartilage tissue cells are cultured on a cell culture substrate coated or supplemented with a temperature-responsive polymer to obtain a chondrocyte sheet, and then the culture solution temperature is equal to or higher than the upper critical solution temperature. Alternatively, it is possible to reduce the number of structural defects by lowering the temperature to the lower critical solution temperature or lower, bringing the cultured layered cell sheet into close contact with the polymer film, and peeling it together with the polymer film, and making it three-dimensionally structured by a predetermined method. It was found that a cell sheet having several functions as a cartilage tissue in vitro and a three-dimensional structure are constructed. Although the method using a cell sheet efficiently regenerates a deficient tissue, the cell sheet must be attached to the affected area, and the transplantation of cells cannot always be completed by a simple operation. There has been a demand for a simple treatment method in which it is sufficient to inject a liquid preparation into a cartilage tissue to be regenerated.

Japanese Patent Application No. 2001-384446 Japanese Patent Application No. 2002-216561 Japanese Patent Application No. 2003-358118 Japanese Patent Application No. 2001-226141 Japanese Patent Application No. 2007-505956

Brittberg et al., New England Journal of Medicine, 331 (14), 889 (1994). Peterson L, 6th Annu. Meet. , American Academic Orthodox Science (1998) Hunziker et al., The Journal of Bone and Joy Surgery, 78-A, 721 (1996).

  In the field of regenerative medicine, transplantation medical technology will develop dramatically when chondrocytes collected from tissues can be rapidly proliferated without being dedifferentiated even if the amount is small, and can be easily injected into the affected area. . An object of the present invention is to provide such a liquid preparation for cartilage regeneration.

In order to solve the above-mentioned problems, the present inventors made hyaluronic acid grafted to a polymer whose hydration power changes in a temperature range of 0 to 80 ° C., and applied it to chondrocytes and synovial cells collected from tissues. The effects were examined in detail. As a result, hyaluronic acid grafted to a polymer whose hydration ability changes in the temperature range of 0 to 80 ° C. expresses the function of chondrocytes present in the gel, and further suppresses inflammation occurring in the transplanted part It was found to have That is, the present invention provides a temperature-responsive chondrocyte-containing liquid formulation that efficiently regenerates cartilage tissue. Moreover, in this invention, the manufacturing method of the formulation is provided. Furthermore, this invention also aims at providing the utilization method of the formulation. By using the temperature-responsive chondrocyte-containing liquid preparation of the present invention, chondrocytes having a very high density and expression can be transplanted, and early tissue regeneration can be performed. At the same time, it was found that inflammation of the transplanted part can be suppressed. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.
That is, the present invention is as follows.
Item 1. A liquid preparation for cartilage tissue regeneration, in which hyaluronic acid grafted to a polymer whose hydration power changes in a physiological saline in a temperature range of 0 to 80 ° C. is dissolved, and 1 × per 1 ml of the preparation A temperature-responsive chondrocyte-containing liquid preparation, which is a dispersion of 10 ⁴ to 1 × 10 ⁷ chondrocytes and suppresses inflammation in a transplanted part.
The temperature-responsive chondrocyte-containing fluid according to claim 1, wherein the hyaluronic acid content in the hyaluronic acid grafted to the polymer whose hydration power changes in the temperature range of 2.0 to 80 ° C is 10 wt% or more. Formulation.
Item 3. The temperature-responsive chondrocyte-containing humoral preparation according to any one of claims 1 and 2, wherein the chondrocytes are human normal chondrocytes.
Item 4. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 3, wherein the chondrocytes are collected from a living tissue.
Item 5. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 4, wherein the polymer whose hydration power changes in a temperature range of 5.0 to 80 ° C is poly (N-isopropylacrylamide). .
Item 6. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 5, wherein the molecular weight of poly (N-isopropylacrylamide) is 4000 to 40000.
Item 7. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 6, wherein the medium contains TGF-β.
Item 8. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 7, which is injected into a cartilage defect.
Item 9. The temperature-responsive chondrocyte-containing liquid preparation according to claim 8, wherein the cartilage defect part is osteochondral defect, cartilage defect, or osteoarthritis.
Item 10. Dissolve chondrocytes by dissolving hyaluronic acid grafted to a polymer whose hydration ability changes at a temperature range of at least 0 to 80 ° C. in a medium or physiological saline under the high hydration state of the polymer. A method for producing a temperature-responsive chondrocyte-containing liquid preparation characterized by comprising:
Item 11. A method for regenerating cartilage, comprising injecting the temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 9 into a cartilage defect portion to regenerate cartilage.
By using the temperature-responsive chondrocyte-containing liquid preparation of the present invention, chondrocytes having a very high density and expression can be transplanted, and early tissue regeneration can be performed. At the same time, it was found that inflammation of the transplanted part can be suppressed. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.

  According to the present invention, it is possible to remarkably enhance the expression of chondrocytes collected from a tissue. Moreover, if the temperature-responsive chondrocyte-containing liquid preparation provided in the present invention is used, inflammation in the transplanted part can be suppressed, and the range of use as a cartilage tissue regenerative medical preparation can be widened.

  It is a figure which shows the result in which the composition ratio in Example 1 and the comparative example 1 influences a phase transition characteristic.   It is a figure which shows the result of having investigated the influence which the hyaluronic acid derivative grafted with poly-N-isopropylacrylamide in Example 2 and Comparative Example 2 has on the gene expression of chondrocytes.   It is a figure which shows the result in which the hyaluronic acid derivative which grafted poly-N-isopropylacrylamide in Example 3 and Comparative Example 3 brings an anti-inflammatory action with respect to a chondrocyte.   In Example 4, the mixture of the hyaluronic acid derivative grafted with poly-N-isopropylacrylamide and the chondrocytes produces results of forming hyaline cartilage subcutaneously in nude mice. (From left, stained with hematoxylin & eosin, alcian blue, and toluidine blue)   In Example 5, it is a figure which shows the result in which the cell growth factor accelerates | stimulates the hypertrophy and maturation of the hyaline cartilage tissue born from the mixture of the hyaluronic acid derivative which grafted poly-N-isopropylacrylamide, and the chondrocyte. (From left, stained with hematoxylin & eosin, alcian blue, and toluidine blue)

  The present invention relates to a liquid preparation for cartilage tissue regeneration, in which hyaluronic acid grafted to a polymer whose hydration power changes in physiological saline in a temperature range of 0 to 80 ° C. Cells are dispersed. At that time, the polymer whose hydration power changes in the temperature range of 0 to 80 ° C. may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. In this case, since the cells to be cultured and peeled are cells, separation is performed in the range of 5 ° C. to 50 ° C., and as the temperature-responsive polymer, poly-Nn-propylacrylamide (of a homopolymer) Lower critical solution temperature 21 ° C), poly-Nn-propylmethacrylamide (27 ° C), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly- N-cyclopropylacrylamide (at 45 ° C), poly-N-ethoxyethylacrylamide (at about 35 ° C), poly-N-ethoxyethylmethacrylamide (at about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (at the same) About 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (about 35 ° C.), poly-N, N-ethylmethyl acetate Ruamido (the 56 ° C.), poly -N, N-diethyl acrylamide (the 32 ° C.), and the like. As monomers for copolymerization used in the present invention, polyacrylamide, poly-N, N-diethylacrylamide, poly-N, N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and salts thereof, polyhydroxyethyl methacrylate, Examples thereof include water-containing polymers such as polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose, carboxymethyl cellulose, and the like, but are not particularly limited. At that time, the molecular weight of the polymer chain is preferably 4000 to 40000, preferably 10,000 to 30000, and more preferably 15000 to 20000. When the molecular weight is less than 4000, the preparation of the present invention cannot be gelled, which is not preferable. On the other hand, when the molecular weight is higher than 40000, the gel strength increases and the operability deteriorates, which is not preferable as the material of the present invention. The present invention refers to a state in which the hydration power of the polymer is increased at the time of manufacturing the preparation and when the preparation is injected into a living body, and the preparation is dissolved in a liquid medium to be described later to improve operability. be able to. On the other hand, after transplantation into a living body, the preparation gels in the affected area and can be retained in the affected area for a long time.

  The hyaluronic acid used in the present invention can be derived from, for example, chicken crown, umbilical cord, lactic acid bacteria, streptococci and the like, but is not particularly limited. The molecular weight is not limited, but the molecular weight of hyaluronic acid is usually 400,000 to 2,000,000. In the present invention, the molecular weight may be within this range, but preferably 700,000 to One of 1,500,000 is preferable, and the most preferable one is 800,000 to 1,000,000. When the molecular weight is larger than 2 million, the operability is deteriorated, which is not preferable for the present invention.

  In the present invention, a polymer in which the hydration power changes in the temperature range of 0 to 80 ° C. and hyaluronic acid bonded by a covalent bond is used. Although the method is not particularly limited, for example, a method of graft polymerization of a monomer that is a raw material of a polymer whose hydration power changes in a temperature range of 0 to 80 ° C. with respect to hyaluronic acid, prepared in advance with respect to hyaluronic acid And a method of graft polymerizing a polymer whose hydration power changes in a temperature range of 0 to 80 ° C., a method of crosslinking hyaluronic acid and a polymer whose hydration power changes in a temperature range of 0 to 80 ° C., and the like. . The hyaluronic acid content in the hyaluronic acid grafted to the obtained polymer whose hydration power changes in the temperature range of 0 to 80 ° C. is not limited, but it is 10 by weight ratio of hyaluronic acid to the total amount of polymer. % Or more, preferably 15% or more, more preferably 25% or more, and most preferably 20% or more. If the content of hyaluronic acid is lower than 10%, the trait of chondrocytes cannot be highly expressed, and the effect of suppressing inflammation of the transplanted part cannot be obtained, which is not preferable as a material used in the present invention. Conversely, it is higher than 25%. The operability deteriorates and is not preferable as the present invention.

Chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, mesenchymal stem cells, adipose-derived cells as suitable cells used in the preparation of the temperature-responsive chondrocyte-containing liquid preparation of the present invention Any one of adipose-derived stem cells, or a mixture of two or more types of cells may be mentioned, but the type is not limited. The mixing ratio at that time is not particularly limited. Although the cell density in the temperature-responsive chondrocyte-containing liquid preparation shown in the present invention varies depending on the cells to be cultured, it is preferable that 1 × 10 ⁴ to 1 × 10 ⁷ cells are dispersed per 1 ml of the preparation. Preferably 5 × 10 ⁴ to 8 × 10 ⁶ , more preferably 1 × 10 ^{5 to} 5 × 10 ⁶ , most preferably 5 × 10 ⁵ to 1 × 10 ⁶ . Things are good. When the cell density is lower than 1 × 10 ⁴ per 1 ml of the preparation, the effect of regenerating cartilage tissue is not obtained, which is not preferable as the present invention. Conversely, when the cell density exceeds 1 × 10 ⁷ , cells and cells aggregate. It becomes easy and is not preferable as the present invention. Furthermore, the cell used in the present invention may be a cell directly collected from a living tissue, a cell directly collected and differentiated by a culture system or the like, or a cell line, but the kind is not limited at all. is not. Furthermore, the origin of the animal of these cells is not particularly limited. For example, mammals such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, chimpanzees, etc. Examples include immunodeficient animals. When the therapeutic cells of the present invention are used for human therapy, it is preferable to use cells derived from humans, pigs, and chimpanzees. The medium for cell culture in the present invention is not particularly limited as long as it is a commonly used medium for cells to be cultured.

  The medium of the present invention is not particularly limited as long as it is used for culturing the cells used in the present invention, but the medium may contain TGF-β as a growth factor. The concentration in the medium is preferably 5 ng / ml or more, preferably 50 ng / ml or more, more preferably 100 ng / ml or more, and most preferably 1000 ng / ml or more. For example, when chondrocytes or cartilage progenitor cells are used as the cells, if the concentration of TGF-β is higher than 5 ng / ml, the cultured cells are efficiently expressed as chondrocytes, which is preferable in the present invention. In the present invention, other medium conditions may be in accordance with conventional methods, and are not particularly limited as long as those commonly used for cultured cells are used. For example, the medium to be used may be a medium to which serum such as known fetal bovine serum (FBS, FCS) is added, or a serum-free medium to which such serum is not added. At that time, the amount of addition is not limited.

  In the method for producing a temperature-responsive chondrocyte-containing liquid preparation in the present invention, hyaluronic acid grafted on the above-described temperature-responsive polymer, cells, and a liquid medium may be mixed. Although the method is not particularly limited, a method in which hyaluronic acid previously grafted to a temperature-responsive polymer is dissolved in physiological saline, and cells suspended in physiological saline are dispersed therein, or cells For example, a method of dispersing itself. In this case, the liquid medium may be a phosphate buffer, but a medium used for cell culture is not preferable. In the present invention, it is preferable to remove the medium as much as possible in the final preparation. In the present invention, the method for removing the medium is not particularly limited, but usually, a method is used in which only cells are collected by a centrifugal separator, membrane filtration or the like and dispersed again in physiological saline.

  The temperature-responsive chondrocyte-containing liquid preparation thus obtained has many characteristics as a preparation for promoting cartilage tissue regeneration. This is specifically shown below by taking chondrocytes as an example. One of them is that the present preparation can enhance the expression of chondrocytes dispersed therein. The reason for this is not always clear, but hyaluronic acid grafted on a temperature-responsive polymer binds to the CD44 receptor on the surface layer of chondrocytes to stabilize chondrocytes and prevent aggregation and dedifferentiation between chondrocytes. I think it is because. In addition, because of its stability, the density of cells in the preparation may be lower than that during normal cell culture. This is also effective when the number of cells available for treatment is limited. That is, by using the temperature-responsive chondrocyte-containing liquid preparation in the present invention, it is possible to provide high-quality cells even in a limited number of cells. Moreover, the hyaluronic acid grafted to the temperature-responsive polymer in the temperature-responsive chondrocyte-containing liquid formulation gels in the affected area, and can protect the affected area from external mechanical action. In general, it is known to transform into fibrocartilage when a mechanical load is applied to the chondrocytes transplanted into the affected area. For example, the cartilage of the knee joint is hyaline cartilage, and is a cell with a different character from fibrocartilage. If the temperature-responsive chondrocyte-containing liquid preparation of the present invention is used for regeneration of knee joint cartilage, not only high quality chondrocytes can be supplied to the affected area, but also transplanted chondrocytes can retain the characteristics of hyaline cartilage. It will be possible. Furthermore, it was also found that hyaluronic acid in this preparation has the effect of suppressing inflammation at the transplanted site. Even if this preparation can be transplanted using an arthroscope or the like, tissue such as the synovium will be damaged by the time the arthroscope reaches the affected area. It has also been found that hyaluronic acid in this preparation has an effect of suppressing inflammation against such inflammation.

  The method of transplanting the temperature-responsive chondrocyte-containing liquid preparation of the present invention into the affected area is not particularly limited, and for example, a method of incising and injecting the affected area, a method of injecting using an endoscope, an arthroscope, etc. In order to reduce the burden on the patient, the latter method is preferable, and the temperature-responsive chondrocyte-containing liquid preparation of the present invention can sufficiently cope with the latter method.

  The cartilage tissue surface of the present invention is not particularly limited as long as it is a cartilage tissue or a bone tissue part, and generally includes articular cartilage, meniscus, intervertebral disc, costal cartilage, nasal septum, auricular bone and the like. The temperature-responsive chondrocyte-containing liquid preparation of the present invention is used for treating an affected part in which a part or all of such cartilage tissue is damaged or missing, or an affected part in which a part of bone tissue is damaged or missing, and is particularly limited. Although not specifically, treatment of arthritis, arthritis, cartilage damage, osteochondral damage, meniscus damage, and intervertebral disc degeneration, especially novel treatment methods for osteoarthritis that were difficult to treat with the prior art It is effective as The method of using the temperature-responsive chondrocyte-containing liquid preparation of the present invention is not particularly limited for such cartilage tissue or bone tissue surface. For example, there is a method of supplementing the temperature-responsive chondrocyte-containing liquid preparation. Can be mentioned. At that time, as described above, the temperature-responsive chondrocyte-containing liquid preparation of the present invention can adhere to the surface of cartilage tissue and bone tissue, which are living tissues, and cannot be obtained from the prior art at all. It is a thing.

  The use of the temperature-responsive chondrocyte-containing liquid preparation shown in the present invention is not limited at all, but for example, osteoarthritis, arthritis, arthropathy, cartilage damage, osteochondral damage, meniscal damage, disc degeneration It is effective for the treatment.

  If the temperature-responsive chondrocyte-containing liquid preparation of the present invention is used, chondrocytes collected from tissues can be proliferated while maintaining their traits. In addition, it becomes possible to suppress inflammation in the transplanted part. These technologies are considered to be extremely effective as regenerative medicine technologies related to tissue regeneration and cell differentiation.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

Poly-N-isopropylacrylamide having a terminal amino group (weight average molecular weight: about 3) by radical polymerization using N-isopropylacrylamide and 2-aminoethanethiol hydrochloride as starting materials and azoisobutyronitrile as a catalyst. .7 × 10 ⁴ ) was synthesized. This amino-terminated poly-N-isopropylacrylamide and hyaluronic acid (weight average molecular weight: about 7.7 × 10 ⁵ ) were mixed at a ratio of 5: 1, and hyaluron grafted with poly-N-isopropylacrylamide by dehydration condensation. Acid derivatives were made. Unreacted substances and low molecular weight components were removed by washing with acetone and dialysis (fraction molecular weight: 25,000). This hyaluronic acid derivative had a sensitive temperature response, and the lower critical solution temperature was about 32 ° C. The lower critical solution temperature was determined from the change in absorbance of the target solution, and was estimated from the inflection point of the slope of the absorbance curve shown at the time of phase transition.

Comparative Example 1

  The amino-terminated poly-N-isopropylacrylamide and hyaluronic acid were mixed at a ratio of 1: 1 to prepare a hyaluronic acid derivative grafted with poly-N-isopropylacrylamide. The temperature responsiveness of the hyaluronsan derivative slowed down, and the lower critical solution temperature increased to about 34.5 ° C.

Normal chondrocytes isolated from rabbit costal cartilage are first cultured and then mixed with the poly (N-isopropylacrylamide) grafted hyaluronic acid derivative prepared in Example 1 under conditions of 37 ° C. and 5% CO ₂ . The cells were cultured for 4 weeks using DMEM / F12 medium containing 10% fetal bovine serum. At this time, 50 μL of a hyaluronic acid derivative solution obtained by grafting 1% concentration of poly-N-isopropylacrylamide to 5.0 × 10 ⁵ chondrocytes was used. Total RNA of chondrocytes was extracted from this cell-containing gel, and the gene expression pattern of cells grown in the gel was analyzed by the reverse transcription-polymerase chain reaction method. As a result, the expression of genes characteristic of normal chondrocytes was confirmed.

Comparative Example 2

Using the same chondrocytes initially cultured in Example 2, an aggregate consisting of only 5.0 × 10 ⁵ chondrocytes was prepared, and other conditions were cultured for 4 weeks under the same conditions as in Example 2. did. After extracting total RNA from this chondrocyte mass, cDNA was synthesized from RNA equivalent to that in Example 2, and the expressed gene was analyzed by the reverse transcription-polymerase chain reaction method. As a result, the expression of genes characteristic of normal chondrocytes was confirmed, but compared to Example 2, the gene expression of transcription factor Sox9 and extracellular matrix aggrecan decreased.

Tumor necrosis factor, which is one of inflammatory cytokines in the medium, after culturing 50 μL of a mixture of 5.0 × 10 ⁵ normal chondrocytes and a hyaluronic acid derivative viscous material grafted with poly-N-isopropylacrylamide for 2 weeks. α was added (10 ⁻⁸ M), and changes in gene expression of proteins related to degradation of extracellular matrix were analyzed in the same manner as in Example 2 24 hours and 72 hours after stimulation. As a result, the expression of matrix metalloproteases MMP3 and MMP13 was remarkably suppressed in chondrocytes cultured in a gel in the derivative, and the ability to suppress the expression was maintained even against stimulation of tumor necrosis factor. That is, it was revealed that the formed cartilage tissue exhibits anti-inflammatory properties.

Comparative Example 3

Cell aggregates consisting of ⁵ normal chondrocytes 5.0 × 10 ⁵ were cultured for 2 weeks, then stimulated with tumor necrosis factor in the same manner as in Example 3, and the protein related to the degradation of extracellular matrix as in Example 3. Changes in gene expression were analyzed. As a result, the expression of MMP3 and MMP13 was detected in chondrocytes of the cell aggregate, and the expression of these genes was increased by stimulation with tumor necrosis factor. That is, when a hyaluronic acid derivative grafted with poly-N-isopropylacrylamide is not used, chondrocytes respond sensitively to inflammatory stimuli and activate the expression of enzymes that degrade their extracellular matrix. It was confirmed.

  150 μL of a mixture of the chondrocytes initially cultured in Example 2 and a hyaluronic acid derivative grafted with poly-N-isopropylacrylamide was injected and implanted subcutaneously into anesthetized nude mice. Two weeks after transplantation, tissues around the transplant were excised from nude mice, paraffin-embedded sections were prepared, and hematoxylin-eosin staining, Alcian blue staining, and toluidine blue staining were performed. As a result, hyaline cartilage-like tissue was newly formed under the skin of nude mice.

  In the same manner as in Example 4, a mixture of chondrocytes and poly-N-isopropylacrylamide grafted hyaluronic acid derivative was prepared, and added so that transforming growth factor-β (TGF-β) was contained at 10 ng / mL. Thereafter, these mixtures were implanted and implanted subcutaneously into nude mice. Thereafter, the tissue around the transplant was observed by histological techniques as in Example 3. As a result, hyaline cartilage-like tissue was newly formed under the skin of nude mice, and the formed cartilage tissue was markedly matured more than that of Example 4.

  According to the present invention, it is possible to remarkably enhance the expression of chondrocytes collected from a tissue. Moreover, if the temperature-responsive chondrocyte-containing liquid preparation provided in the present invention is used, inflammation in the transplanted part can be suppressed, and the range of use as a cartilage tissue regenerative medical preparation can be widened.

Claims (11)

A liquid preparation for cartilage tissue regeneration, in which hyaluronic acid grafted to a polymer whose hydration power changes in a physiological saline in a temperature range of 0 to 80 ° C. is dissolved, and 1 × per 1 ml of the preparation A temperature-responsive chondrocyte-containing liquid preparation, which is a dispersion of 10 ⁴ to 1 × 10 ⁷ chondrocytes and suppresses inflammation in a transplanted part. The temperature-responsive chondrocyte-containing liquid preparation according to claim 1, wherein the hyaluronic acid content in the hyaluronic acid grafted to the polymer whose hydration power changes in a temperature range of 0 to 80 ° C is 10 wt% or more. The temperature-responsive chondrocyte-containing humoral preparation according to any one of claims 1 and 2, wherein the chondrocytes are human normal chondrocytes. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 3, wherein the chondrocytes are collected from a living tissue. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 4, wherein the polymer whose hydration power changes in a temperature range of 0 to 80 ° C is poly (N-isopropylacrylamide). The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 5, wherein the molecular weight of poly (N-isopropylacrylamide) is 4000 to 40000. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 6, wherein the medium contains TGF-β. The temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 7, which is injected into a cartilage defect. The temperature-responsive chondrocyte-containing liquid preparation according to claim 8, wherein the cartilage defect part is osteochondral defect, cartilage defect, or osteoarthritis. Dissolve chondrocytes by dissolving hyaluronic acid grafted to a polymer whose hydration ability changes at a temperature range of at least 0 to 80 ° C. in a medium or physiological saline under the high hydration state of the polymer. A method for producing a temperature-responsive chondrocyte-containing liquid preparation characterized by comprising: A method for regenerating cartilage, comprising injecting the temperature-responsive chondrocyte-containing liquid preparation according to any one of claims 1 to 9 into a cartilage defect portion to regenerate cartilage.